At physiological temperature and pH, DNA molecules assume the native, duplex form. Intramolecular hydrogen bonds and stacking interactions are responsible for the maintenance of base pairing between complementary DNA strands. If base pairing is disrupted (e.g. through thermal melting) the native molecule assumes a randomly coiled, single-stranded form. Once denatured, the capacity for the DNA to reanneal at complementary regions depends upon nucleotide sequence homology. The localization of specific DNA sequences is achieved through hybridization of a labeled complementary probe to the DNA sequence of interest, under conditions which favor localized duplex formation. Restriction endonuclease cleavage of large duplex DNAs into small fragments which can be sized by gel electrophoresis, in coordination with hybridization of probes to their complementary DNA sequences within these fragments, has enabled the mapping, sizing, and detection of specific genes within these subregions.
The localization of specific DNA sequences within restriction fragments is accomplished usually through a process known as Southern Transfer (Southern, E. M., J. Mol. Biol. 98:503-517 (1975)). In this technique DNA fragments are electrophoresed on an agarose gel, denatured, and transferred to a nitrocellulose filter. The immobilized DNA is then probed with .sup.32 P-labeled DNA or RNA. Autoradiography is employed to visualize DNA fragments to which the hybridized probe is complementary. Since the nitrocellulose is a porous, rigid support for DNA, the rate of hybridization between the probe and the DNA sequence is slow since only one component of the reaction is allowed to search for its complement. Furthermore, because .sup.32 P has an affinity for nitrocellulose, the filter must be washed thoroughly to rid it of any loosely bound probe in an attempt to reduce background noise.